The long-term goal of the proposed research is to disentangle the contributions of the forces that govern the evolution of the Drosophila genome. In particular, this project focuses on the role of mutation rate variation in patterns of molecular evolution in Drosophila melanogaster. Since mutational generation of novel allelic variants is the first step of the molecular evolutionary process, understanding the effects of heterogeneity in mutation rate on the evolution of coding and noncoding sequences will contribute greatly to the understanding of molecular evolutionary dynamics of the D. melanogaster genome. To address this question, I will first establish an appropriate neutral mutational model for DNA sequence evolution and then use this null model for hypothesis-driven testing to investigate patterns of evolution in functional portions of the genome. Specifically, I propose to characterize the neutral mutational spectrum in the Drosophila melanogaster genome by surveying polymorphism in unconstrained dead-on arrival (DOA) non-long-terminal-repeat (non-LTR) retrotransposable elements. By comparing the polymorphism and divergence spectra of these sequences I can test whether these sequences are evolving in a manner that is consistent with neutrality and potentially validate the approach of using substitutional patterns from unconstrained sequences to infer the underlying mutational process. Beyond characterizing the mutational spectrum and validating the neutrality of DOA sequences, these polymorphism and divergence data can inform our understanding of the role of single-nucleotide mutations in generating observed substitutional patterns at adjacent coding and noncoding regions, and will therefore be applied in two additional contexts. I will first test whether mutation rate variation alone is sufficient to explain the extensively documented heterogeneity in rates and patterns of substitution in this organism by comparing polymorphism and divergence spectra from different parts of the genome previously documented to have significantly different substitutional profiles. Second, I will use these polymorphism and divergence data from the DOA elements to evaluate the contribution of mutation rate variation and selection to rates of evolution in adjacent coding and noncoding sequences in the D. melanogaster genome. This proposed research is relevant to public health in that the findings will deepen our understanding of the forces that shape genome evolution, and will contribute to the development of tools for comparative functional annotation of genomes of organisms as diverse as insects, mammals, and pathogens.